Alzheimer's disease (AD) is a degenerative disorder of the CNS found primarily among the elderly that is characterized by progressive memory loss. Among the hallmarks of the disease is the extracellular deposition of neuritic plaques of amyloid-p peptide (AP). Familial forms of AD (FAD) are often associated with mutations in the genes encoding presenilin (PS) proteins. Several groups have noted that FAD-linked PS mutations sensitize neurons to apoptosis due to Ap. The goal of this proposal is to gain insight into the interaction between AP and PS mutations in a cell culture model of AD. The experiments proposed will address two major aspects of intracellular signaling: (a) Ca2+ dynamics and (b) pro-apoptotic signal transduction cascades. Evidence suggests that Ap-induced cell death is due, at least in part, to the disruption of intracellular Ca2+ homeostasis. In addition, FAD-linked PS mutations have been associated with increased Ca2+ release from intracellular stores. These exaggerated Ca2+ responses have played a central role in the development of the "Ca2+ overload" hypothesis which states that ER Ca2+ stores are overfilled, and this overfilling is a central event in AD. More recent work has called the "Ca2+ overload" hypothesis into question. The experiments proposed seek to directly evaluate the "Ca2+ overload" hypothesis by looking at cytosolic and store Ca2+ levels in SY5Y neuroblastoma cells expressing mutant presenilins upon challenge with Ca2+ channel agonists or Ap. Parallel studies will be conducted with Ca2+ channel antagonists in order to evaluate the specificity of the agonist response. The disruption of intracellular Ca2+ homeostasis is part of a cascade of events that lead to apoptosis following exposure to Ap. We are interested in the role of the unfolded protein response (UPR) in mediating the pro-apoptotic effects of Ap. Therefore, the induction and/or activation of several UPR associated proteins will be assessed in mutant PS-expressing SY5Y cells. It has been suggested that the differential effects of PS mutations on Ca2+ homeostasis could influence the cellular response to death stimuli, such as exposure to Ap, thus promoting or delaying disease onset and progression. Collectively, the data from the proposed studies will allow us to assess the validity of this suggestion.